GSC Biological and Pharmaceutical Sciences, 2020, 13(02), 266-272
Available online at GSC Online Press Directory
GSC Biological and Pharmaceutical Sciences
e-ISSN: 2581-3250, CODEN (USA): GBPSC2
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Corresponding author: Obou Constantin Okou
Department of Biochemistry and Microbiology, Agroforestry Training and Research Unit, Jean Lorougnon Guédé University, Côte
Copyright © 2020 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.
(RE SE AR CH AR T I CL E)
Evaluation of the hemogram of Oryctolagus cuniculus envenomus
Obou Constantin Okou 1, 4. *, N’guessan Emmanuel Assemian 2, Kouadio Bernard Allali 3, Guy Childeric Bingo
1 and Allico Joseph Djaman 4
1 Department of Biochemistry and Microbiology, Agroforestry Training and Research Unit, Jean Lorougnon Guédé
University, Côte d'Ivoire.
2 Department of Biology and Tropical Ecology, Environmental Training and Research Unit, Jean Lorougnon Guédé
University, Côte d'Ivoire.
3 Environment and Health Department, Entomology and Herpetology Unit, Pasteur Institute of Côte d'Ivoire.
4 Department of Biology-Health, Biosciences Training and Research Unit, Félix Houphouët-Boigny University Abidjan,
Publication history: Received on 15 November 2020; revised on 25 November 2020; accepted on 27 November 2020
Article DOI: https://doi.org/10.30574/gscbps.2020.13.2.0384
The overall objective of this study was to evaluate the hemolysing action of Naja nigricollis venom on rabbit blood. To
carry out this study, three batches of three rabbits were formed with two control batches and one experimental batch.
Each control lot is composed of three rabbits (males or females) while the experimental lot is composed of two males
and one female. Each rabbit from the control lots was separately collected in the purple tube (EDTA) and transported
to the laboratory for analysis. The rabbits from the experimental batch were also collected distinctly a few minutes after
the injection of the venom of Naja nigricollis for the analysis of haematological parameters. However, before the analysis
of the hematological parameters of the rabbits from the control and experimental batches, an in vitro hemolysis test of
Naja nigricollis venom was performed to verify its hemolysing power. The results showed that Naja nigricollis venom
has a dose-dependent in vitro hemolysing power. As for the haemogram, it revealed that the venom of Naja nigricollis
has a decreasing effect on blood cells (red and white blood cells), on haemoglobin and on haematocrit, and an elevation
on MGVs thus promoting anaemia.
Keywords: Venom; Naja nigricollis; Haemogram
Envenimation is the introduction into the body of a toxic substance, venom, due to the bite of a snake, the sting of a
scorpion, a wasp, etc. The most dangerous is that caused by snake bites , . Indeed, venom contains a complex
mixture of enzymes, peptides and proteins of low relative molecular weight, with specific chemical and biological
activities, which can lead to death from neurological and/or hematological disorders , . This case of envenimation
is a problem affecting the five continents of the world. Globally, the annual incidence of snake bites exceeds six million
, , . However, Africa records more than one million bites causing 600,000 cases of envenomation. Also in Africa,
nearly 250,000 patients are treated, nevertheless, there are more than 20,000 deaths , .
In Africa, generally speaking, the venomous snakes responsible for all these disasters are mainly Viperidae and Elapidae.
Viperidae (vipers) are the most widespread venomous species and do the majority of damage while Elapidae are the
GSC Biological and Pharmaceutical Sciences, 2020, 13(02), 266–272
most dangerous snakes because of the high toxicity of their venom . It is classic to oppose cobraic poisoning,
essentially neurotoxic, and viperine poisoning, dominated by necrosis and hemorrhagic syndromes. In practice, this
distinction must be qualified . In fact, the diversity of substances, contained in the venoms of the species of these two
families of snakes, vary according to the species and even between individuals of the same species, which makes certain
species exceptional by the action of their venom. Among these exceptions is the venom of Naja nigricollis, one of the
most dangerous and representative species of Elapidae in Africa. Its venom contains cytoxins that target certain blood
cells and those of certain organs such as the heart. Its action on these organs creates dysfunctions whose effects
associated with those of these neurotoxins could have a serious impact on the breathing of the victim and lead to his
death , .
In fact, the haematological parameters or haemogram are made up of blood figurative elements (blood cells) divided
into red lineage cells (red blood cells, reticulocytes), white lineage cells (lymphocytes, granulocytes, monocytes) and
platelets. Under physiological conditions, the proportion of each cell type is within a range determined for each species.
The absolute or relative amount of the different cell types may vary following exposure to a toxicant or under
pathological conditions . According to the same author, hematological toxicity is expressed by an increase or
decrease in the number of peripheral blood cells of one or more cell lines. These cytopenias have various consequences:
anemic syndrome, hemorrhagic, infectious by immunosuppression, etc.... The origin of this change in the blood count
may be central by spinal cord injury or peripheral by immune destruction or not. The objective of this study was to
evaluate the haemogram of a rabbit that had become envenomized.
2. Material and methods
2.1. Animal material
For this study, nine rabbits including five males and four females of Hyplus breed, aged two and a half months were
purchased from a breeder in the locality of Daloa (Côte d’Ivoire). After the acclimatization period the weight of the
rabbits varied between 1.45 and 2.4 kg. Beside this animal model, the venom of Naja nigricollis (spitting cobra) was also
used and supplied by the Pasteur Institute of Adiopodoumé (Côte d’Ivoire).
2.2. In vitro hemolysis test method
Two control lots (lot 1 and lot 2) were established for this test. Lot 1 consists of three males and lot 2 consists of three
females. Among these control lots, a blood sample was taken from one of them in order to perform the in vitro hemolysis
test of Naja nigricollis venom. To perform this test, 10 tubes were used, including a control tube and 9 experimental
tubes. The stock solution was prepared in tube 1 by dissolving 1.6 mg of venom crystals in 1 mL of physiological water.
In the remaining 9 tubes (tubes 2 to 10) a volume of 0.5 mL of physiological water was added. The venom concentration
ranges were prepared using the double dilution technique of geometric ratio 1/2. It consisted in taking 0.5 mL of the
stock solution (tube 1) and transferring it to 0.5 mL of physiological water from tube 2 and homogenizing it. This
procedure was repeated up to tube 9. From tube 9, a volume of 0.5 mL was collected and subsequently discarded. Thus,
concentrations in the tubes ranged from 1.6 mg/mL to 6.26.10-3 mg/mL. To these 9 experimental tubes and to the
control tube (tube 10), 5 drops of rabbit whole blood was added and homogenized manually. After homogenization, all
preparations were incubated at room temperature for 30 to 40 minutes for microscopic observation. This observation
was carried out at magnification 40 (X40). For this purpose, the preparations in tubes 1 (SS); 2; 3 and tube 10 (control
tube) were diluted distinctly to 1/5th. Then each dilution was spread between slide and slide by putting the dilution of
tube 10 (control) and an experimental dilution (e.g. tube 1). This last operation was also performed for tube 2 and tube
2.3. Method of blood collection
In general, blood samples were taken from the short saphenous vein and/or the femoral vein. The restraint method was
performed by three people. The areas where these veins were located were previously exposed with a pair of scissors.
The vacutainers into which the needles were inserted allowed the sampling to be carried out using the purple tubes
(EDTA). The resulting tubes were stored in a cooler containing ice and then transported to the laboratory for analysis.
2.4. Method of carrying out experimental tests
For the experimental tests, a quantity of 2 mg of venom crystals was dissolved in 0.5 mL of physiological water in order
to have a concentration of 4 mg/mL. This is the concentration that was injected into the rabbits. Indeed, according to
, the median lethal dose for a 2 kg rabbit is 2 mg/kg body weight in intramuscular injection.